Liposomes are lipid bilayer vesicle nanoparticles which have attracted great interest as drug delivery vehicles (Kikuchi, H. et al. (1999) “Gene delivery using liposome technology,” J. Conto. Rel. 62:269-277; Templeton et al. (1997) “Improved DNA: Liposome complexes for increased systemic delivery and gene expression,” Nat. Biotechnol. 15:647-652; Abraham. S. et al. (2005) “The liposomal formulation of doxorubicin,” Methods Enzymol. 391:71-97; Andresen, T. L. et al. (2005) “Advanced strategies in liposomal cancer therapy: Problems and prospects of active and tumor specific drug release,” Prog. Lipid Res. 44:68-97; Ramachandran, S. et al. (2006) “Nanoliposomes for Cancer Therapy: AFM and Fluorescence imaging of Cisplatin Encapsulation, Stability, Cellular Uptake and Toxicity,” Langmuir 22:8156-8162; Zamboni, W. C. (2005) “Liposomal Nanoparticle, and Conjugated Formulations of Anticancer Agents,” Clin. Cancer Res. 11:8230-8234). Encompassing the ability to encapsulate aqueous solutions within their core, isolate lipophilic compounds within their lipid bilayer, and support tailored surface chemistries for targeted delivery, liposomes are versatile, multifunctional nanoparticles with numerous applications including drug delivery for cancer treatment, antibiotics, and anesthetic compounds (Allen, T. M. & Cullis, P. R. (2013) “Liposomal drug delivery systems: From concept to clinical applications,” Adv. Drug Delivery Rev. 65:36-48; Fenske, D. B. et al. (2008) “Liposomal nanomedicines: an emerging field,” Toxicologic Pathology 36:21-29).
Due to their ability to increase the drug loading capacity by an order of magnitude or more compared to other delivery methods (Gu, F. X. et al. (2007) “Targeted nanoparticles for cancer therapy,” Nano Today 2:14-21) and protect their payloads from metabolic activity and early excretion, liposomal drugs provide increased therapeutic indices while minimalizing the damaging side effects of their non-encapsulated counterparts (Immordino, M. L. et al. (2006) “Stealth liposomes: review of the basic science, rationale, and clinical applications, existing and potential,” International J. Nanomedicine, 1(3):297-315). As such, liposome encapsulated drugs have had an immense impact in oncology, with long circulating liposomes providing preferential extravasation from tumor vessels (Park, J. W. et al. (2002) “Future directions of liposome- and immunoliposome-based cancer therapeutics,” Semin. Oncol. 31:196-205).
Liposome-encapsulated drugs have exhibited potent activity against a wide range of cancers including breast, ovarian, uterine, and other solid tumors (Gu, F. X. et al. (2007) “Targeted nanoparticles for cancer therapy,” Nano Today 2:14-21; Patri, A. K. et al. (2002) “Dendritic polymer macromolecular carriers for drug delivery,” Current Opinion in Chemical Biology 6:466-471; Wang, A. Z. et al. (2012) “Nanoparticle Delivery of Cancer Drugs,” Annu. Rev. Med. 63:185-98; Brannon-Peppas, L. & Blanchette, J. O. (2012) “Nanoparticle and targeted systems for cancer therapy,” Adv. Drug Deliv. Rev. 64:206-212; Gabizon, A. & Martin, F. (1997) “Polyethylene glycol-coated (pegylated) liposomal doxorubicin. Rationale for use in solid tumors.” Drugs 54:15-21; Krishna, R. & Mayer, L. D. (1997) “Liposomal doxorubicin circumvents PSC 833-free drug interactions, resulting in effective therapy of multidrug resistant solid tumors,” Cancer Res. 57:5246-53). Liposome delivery systems have also had an impact in vaccinology (Glück, R. (1995) “Liposomal presentation of antigens for human vaccines,” Pharm. Biotechnol. 6:325-45), ophthalmology (Ebrahim, S. et al. (2005) “Applications of liposomes in ophthalmology,” Surv. Ophthalmol. 50:167-82), pulmonology (Schreier, H. et al. (1993) “Pulmonary delivery of liposomes,” J. Control. Release 24:209-223), and numerous other pathologies (Moghimi, S. M. & Szebeni, J. (2003) “Stealth liposomes and long circulating nanoparticles: critical issues in pharmacokinetics, opsonization and protein-binding properties,” Prog. Lipid Res. 42:463-478; Tazina, E. V. et al. (2011) “Qualitative and quantitative analysis of thermosensitive liposomes loaded with doxorubicin,” Pharm. Chem. J. 46:54-59).
Tumor microvasculature is porous, with pore diameters large enough to allow nanoparticles smaller than several hundred nanometers to migrate into the extravascular space, providing a mechanism for liposome-encapsulated drugs to concentrate within tumors (Abraham. S. et al. (2005) “The liposomal formulation of doxorubicin,” Methods Enzymol. 391:71-97). Using this feature, liposomal anthracyclines have shown reduced toxicity compared with conventional delivery methods, while providing efficacies comparable with their conventional counterparts (O'Shaughnessy, J. (2003) “Liposomal Anthracyclines for Breast Cancer: Overview,” Oncologist 8:1-2). For example, Doxil®, the first FDA-approved nanoparticle drug, comprising the anthracycline antibiotic doxorubicin in PEGylated ˜100 nm liposomes (Barenholz, Y. C. (2012) “Doxil®—The First FDA-Approved Nano-Drug: Lessons Learned,” J. Controlled Release 160:117-134), is widely used as a chemotherapeutic for treatment of a range of recurrent cancers, and there are various other liposomal drugs approved for clinical use (Chang, H. I. & Yeh, M. K. (2012) “Clinical development or liposome-based drugs: formulation, characterization, and therapeutic efficacy,” International J. of Nanomedicine 7:49-60; Wagner, V. et al. (2006) Nat. Biotechnol. 24:1211-7), with many more formulations in clinical trials.
Vesicle size and polydispersity are key parameters impacting the therapeutic index of liposomal drugs. Smaller liposomes are known to exhibit slower blood clearance rates, thereby increasing drug bioavailability (Chang, H. I. & Yeh, M. K. (2012) “Clinical development or liposome-based drugs: formulation, characterization, and therapeutic efficacy,” International J. of Nanomedicine 7:49-60; Litzinger, D. C. et al. (1994) “Effect of liposome size on the circulation time and intraorgan distribution of amphipathic poly(ethylene glycol)-containing liposomes,” Biochim. Biophys. Acta 1190:99-107). Various attempts have been made to investigate the impact of liposome size on cell uptake, intracellular transport and fate, and overall biodistribution for vesicles in the ˜100 to ˜1,000 nm range (Ramachandran, S. et al. (2006) “Nanoliposomes for Cancer Thereapy: AFM and Fluorescence imaging of Cisplatin Encapsulation, Stability, Cellular Uptake and Toxicity,” Langmuir 22:8156-8162; Kelly, C. et al. (2011) “Targeted Liposomal Drug Delivery to Monocytes and Macrophages,” J. Drug Delivery 2011, 727241; Ahsan, E. et al. (2002) “Targeting to macrophages: role of physiochemical properties of particulate carriers—liposomes and microspheres—on the phagocytosis by macrophages,” J. Controlled Release 79:29-40; Epstein-Barash, H. et al. (2010) “Physicochemical parameters affecting liposomal bisphosphonates bioactivity for restenosis therapy: internalization, cell inhibition, activation of cytokines and complement, and mechanism or cell death,” J. Controlled Release 146:182-195; Takano, S. et al. (2003) “Physicochemical properties of liposomes affecting apoptosis induced by cationic liposomes in macrophages,” Pharmaceutical Research 20:962-968; Pollock, S. et al. (2010) “Uptake and trafficking of liposomes to the endoplasmic reticulum,” The FASEB 24:1866-1878). In some studies, liposomes larger than 300 nm were not effectively taken up by cells in vitro, while smaller 100 nm liposomes exhibited rapid endocytosis (Ramachandran, S. et al. (2006) “Nanoliposomes for Cancer Therapy: AFM and Fluorescence imaging of Cisplatin Encapsulation, Stability, Cellular Uptake and Toxicity,” Langmuir 22:8156-8162). In other studies, 100 nm liposomes were found to maximize uptake into monocytes and macrophages compared with larger vesicles (Kelly, C. et al. (2011) “Targeted Liposomal Drug Delivery to Monocytes and Macrophages,” J. Drug Delivery 2011, 727241; Ahsan, E. et al. (2002) “Targeting to macrophages: role of physicochemical properties of particulate carriers—liposomes and microspheres—on the phagocytosis by macrophages,” J. Controlled Release 79:29-40; Epstein-Barash, H. et al. (2010) “Physicochemical parameters affecting liposomal bisphosphonates bioactivity for restenosis therapy: internalization, cell inhibition, activation of cytokines and complement, and mechanism or cell death,” J. Controlled Release 146:182-195; Takano, S. et al. (2003) “Physicochemical properties of liposomes affecting apoptosis induced by cationic liposomes in macrophages,” Pharmaceutical Research 20:962-968). In the case of liposomal doxorubicin, higher tumor uptake has been observed together with significantly lower uptake by healthy tissues when the mean vesicle size was reduced from 100 nm to 75 nm (Cuia, J. et al. (2007) “Direct comparison of two pegylated liposomal doxorubicin formulations: is AUC predictive for toxicity and efficacy?” J. Controlled Release 118:204-215). As such, questions remain about the impact of liposome size on cell uptake and internalization for vesicles smaller than about 100 nm, in part due to limitations imposed by most existing liposome preparation techniques.
Thus, a challenge to liposomal drug delivery technologies has been the effective control over vesicle size during synthesis. The ideal size for cancer-targeting liposomal nanomedicines is commonly believed to be about 100 nm, which is thought to be large enough to provide a high drug payload volume while small enough to pass through leaky endothelial junctions in tumor tissues (Fenske, D. B. et al. (2008) “Liposomal nanomedicines: an emerging field,” Toxicologic Pathology 36:21-29). However, such view ignores the increasing use of liposomes for lipophilic drug encapsulation within the vesicle membrane where loading efficiency scales inversely with liposome size, and also ignores the impact of vesicle size on key parameters affecting drug efficacy and safety, including cellular uptake, cellular fate, and overall biodistribution. Relationships between such key characteristics and liposome size are not fully understood for nanoparticles below 100 nm, in large part because many conventional bulk synthesis techniques yield relatively large and polydisperse liposome populations that render detailed size-dependent studies difficult.
Another challenge hampering liposomal delivery systems has been the development of effective methods for loading high concentrations of therapeutic agent(s) or drug into lipid vesicles. Increased drug-to-lipid ratio (D/L) is a highly desirable attribute for liposome delivery systems, given in vivo toxicity is inversely related to D/L (Mayer, L. D. et al. (1989) “Influence of Vesicle Size, Lipid Composition, and Drug-to-Lipid Ratio on the Biological Activity of Liposomal Doxorubicin in Mice” Cancer Res. 49:5922-5930). For example, nanoparticle delivery systems can enhance the therapeutic index of anti-cancer agents by increasing drug concentration in tumor cells. The increased drug concentration facilitated by nanoparticle delivery is the result of enhanced nanoparticle permeability and retention in tumor tissues (Matsumura, Y. & Maeda. H. (1986) “A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent SMANCS,” Cancer Res 6:193-210; Maeda, H. (2010) “Tumor-selective delivery of macromolecular drugs via the EPR effect: background and future prospects,” Bioconjugate Chemistry 21:797-802), together with the use of molecular targeting strategies that enhance tumor cell uptake (Moses, M. A., Brem. H. & Langer. R. (2003) “Advancing the field of drug delivery: taking aim at cancer,” Cancer Cell 4:337-341; Liu, Y. et al. (2007) “Nanomedicine for drug delivery and imaging: a promising avenue for cancer therapy and diagnosis using targeted functional nanoparticles,” International J. Cancer 120:2527-2537; Cho, K. et al. (2008) “Therapeutic nanoparticles for drug delivery in cancer,” Clin. Cancer Research 14:1310-1316).
A variety of liposomal drug synthesis techniques have been reported (Otake. K. et al. (2006) “Preparation of liposomes using an improved supercritical reverse phase evaporation method,” Langmuir The ACS Journal of Surfaces and Colloids 22:2543-2550; Uhumwangho, M. U. & Okor, R. S. (2005) “Current trends in the production and biomedical applications of liposomes: a review,” Sciences New York 4:9-21; Jiskoot, W. et al. (1986) “Preparation of liposomes via detergent removal from mixed micelles by dilution. The effect of bilayer composition and process parameters on liposome characteristics,” Pharmaceutisch Weekblad Scientific Edition 8:259-265; Szoka, F. & Papahadjopoulos, D. (1978) “Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse phase evaporation,” Proceedings of the National Academy of Sciences of the United States of America 75:4194-4198; Meure, I. A. et al. (2008) “Conventional and Dense Gas Techniques for the Production of Liposomes: A Review,” Aaps Pharmscitech 9:798-809).
Conventional liposome preparation is based on demanding bulk-scale processes which include a variety of traditional methods including ethanol injection, reverse-phase evaporation, detergent depletion, emulsification, supercritical phase formation, membrane extrusion, thin-film hydration, rapid solvent exchange, which all require post-processing steps such as sonication or membrane extrusion to regulate the size and reduce the polydispersity of the final population of liposomes (Jesorka, A. & Orwar, 0. (2008) “Liposomes: technologies and analytical applications” Annu. Rev. Anal. Chem. (Palo Alto. Calif. 1:801-32). In addition, such techniques require further processing steps for drug encapsulation, membrane functionalization, purification, and concentration. Conventional bulk methods are therefore cumbersome, time consuming, and labor intensive, and result in liposomal nanomedicines with limited shelf life due to drug leakage and lipid degradation. Moreover, even after repeated processing steps utilizing bulk techniques, such as sequential membrane extrusion or size exclusion chromatography, the resulting vesicles tend to be relatively large (>100 nm) and polydisperse. For example, when performing 6-step membrane extrusion to reduce liposome size and polydispersity, relative standard deviations above 50% are observed (Berger, N. et al. 2001) “Filter extrusion of liposomes using different devices: comparison of liposome size, encapsulation efficiency, and process characteristics,” International Journal of Pharmaceutics 223:55-68). In addition, conventional bulk synthesis and encapsulation methods often lead to significant agent loss with waste that can approach 98% for hydrophilic drug compounds (Lasic, D. D. (1998) “Novel applications of liposomes,” Trends in Biotechnology 16:307-321; Nagayasu, A. et al. 1999) “The size of liposomes: a factor which affects their targeting efficiency to tumors and therapeutic activity or liposomal antitumor drugs,” Advanced Drug Delivery Reviews 40:75-87).
Because conventional bulk synthesis yields relatively large and polydisperse liposomes, detailed size-dependent behaviors for smaller vesicles have proven difficult or impossible to study. As a result, studies of size-dependent behaviors have largely focused on the use of inorganic nanoparticles such as gold (Chithrani, B. D. et al. (2006) “Size and Shape Dependence of Nanoparticles on Cellular Uptake,” Nano 668:662-668; Shan, Y. et al. (2009) “Size-dependent endocytosis of single gold nanoparticles,” Chemical Communications 47:8091-8093; Zhang, S. et al. (2009) “Size-Dependent Endocytosis of Nanoparticles,” Advanced materials Deerfield Beach Fla. 21:419-424; Cho, E. C. et al. (2011) “Cellular uptake of gold nanoparticles,” Cancer Cell 6:385-391), carbon (Jin, H. et al. (2009) “Size-dependent cellular uptake and expulsion of single-walled carbon nanotubes: single particle tracking and a generic uptake model for nanoparticles,” ACS nano 3:149-158), iron oxide (Huang, J. et al. (2010) “Effects of nanoparticle size on cellular uptake and liver MRI with polyvinylpyrrolidone-coated iron oxide nanoparticles,” ACS nano 4:7151-7160), and silica (Kumar, S. et al. (2012) “Size Dependent Interaction of Silica Nanoparticles with Different Surfactants in Aqueous Solution,” Langmuir 28(25):9288-9297, which materials can be synthesized with relatively tight control over size and with low polydispersity. However, because surface properties of such inorganic nanoparticles are entirely different from liposomes, which closely mimic the native cell walls across which endocytosis occurs, the results of such studies cannot be translated and is thus not relevant to liposomal drug delivery.
Microfluidic technologies have been attempted to alleviate some of the shortcomings of conventional bulk-scale liposome production methods (Andar, A. U. et al. (2014) “Microfluidic Preparation of Liposomes to Determine Particle Size Influence on Cellular Uptake Mechanisms” Pharm. Res. 31:401-13; Hood, R. R. et al. (2014) “Microfluidic-Enabled Liposomes Elucidate Size-Dependent Transdermal Transport,” PLoS One 9:e92978). Controlled liposome formation utilizing a microfluidic hydrodynamic flow-focusing (MHF) technique substantially decreases size variance, with fewer processing steps. Compared to conventional bulk-scale techniques, traditional MHF methods provide nanoparticles with enhanced properties including adjustable, narrowly distributed diameters (Jahn, A. et al. (2004) “Controlled Vesicle Self-Assembly in Microfluidic Channels with Hydrodynamic Focusing,” J. Am. Chem. Soc. 126:2674-2675; Jahn, A. et al. (2007) “Microfluidic Directed Self-Assembly of Liposomes of Controlled Size,” Langmuir 23:6289-6293; Jahn, A. et al. (2008) “Preparation of nanoparticles by continuous-flow microfluidics,” J. Nanoparticle Res. 10:925-934; Jahn, A. et al. (2010) “Microfluidic mixing and the formation of nanoscale lipid vesicles,” ACS nano 4:2077-2087), and tunable physiochemical properties (Hood, R. et al. (2013) “Microfluidic synthesis of PEGylated and folate receptor-targeted liposomes,” Pharm. Res. 30:1597-607). However, realtively low throughput has been achieved by traditional microfluidic systems, which has constrained MHF methods for use in bulk production (e.g., such as for large scale in vivo studies and preclinical trials where larger volumes and higher concentrations are required). Thus, sufficient throughput and nanoparticle concentration have not been achieved utilizing traditional microfluidic techniques.
Accordingly, there is a need for microfluidic methodologies and systems that overcome some or all of the above-noted limitations and/or disadvantages.